Chimeric Antigen Receptor-T Cells with 4-1BB Co-Stimulatory Domain Present a Superior Treatment Outcome than Those with CD28 Domain Based on Bioinformatics.

BACKGROUND: The second-generation CD19-chimeric antigen receptor (CAR)-T co-stimulatory domain that is commonly used in clinical practice is CD28 or 4-1BB. Previous studies have shown that the persistence of CAR-T in the 4-1BB co-stimulatory domain appears to be longer. METHODS: The expression profile data of GSE65856 were obtained from GEO database. After data preprocessing, the differentially expressed genes (DEGs) between the mock CAR versus CD19-28z CAR T cells and mock CAR versus CD19-BBz CAR T cells were identified using the limma package. Subsequently, functional enrichment analysis of DEGs was performed using the DAVID tool. Then, the protein-protein international (PPI) network of these DEGs was visualized by Cytoscape, and the miRNA-target gene-disease regulatory networks were predicted using Webgestal. RESULTS: A total of 18 common DEGs, 6 CD19-28z specific DEGs and 206 CD19-BBz specific DEGs were identified. Among CD19-28z specific DEGs, down-regulated PAX5 might be an important node in the PPI network and could be targeted by miR-496. In CD19-BBz group, JUN was a hub node in the PPI network and involved in the regulations of miR520D - early growth response gene 3 (EGR3)-JUN and mi-R489-AT-rich interaction domain 5A (ARID5A)-JUN networks. CONCLUSION: The 4-1BB co-stimulatory domain might play in important role in the treatment of CAR-T via miR-520D-EGR3-JUN and miR489-ARID5A-JUN regulation network, while CD28 had a negative effect on CAR-T treatment.

Knockdown of REGγ inhibits the proliferation and migration and promotes the apoptosis of multiple myeloma cells by downregulating NF-κB signal pathway.

OBJECTIVES: This study aimed to evaluate the effects of REGγ knockdown on the proliferation, apoptosis and migration of multiple myeloma (MM) cells, and reveal the potential regulatory mechanisms. METHODS: The expression of REGγ on myeloma cells of 28 MM patients was detected by Western blot. shRNA-REGγ-1 and shRNA-REGγ-2 were constructed to downregulate REGγ in RPMI-8226 cells. The proliferation, apoptosis and migration of transfected cells were analyzed by Cell Counting Kit 8 (CCK8), flow cytometry and transwell chamber, respectively. The expression of phosphorylated p65 (p-p65), p65, NF-kappa-B inhibitor ε (IkBε), matrix metalloproteinase 2 (MMP2), B-cell lymphoma xL (Bcl-xL) and X-linked inhibitor of apoptosis protein (XIAP) in transfected cells was detected by Western blot. Using cycloheximide (CHX), the half-life period of IkBε was detected by Western blot. RESULTS: The expression of REGγ was positive in myeloma cells. The proliferation and migration of RPMI-8226 cells were significantly inhibited by shRNA-REGγ-1/shRNA-REGγ-2, while the apoptosis rates were significantly increased (p < 0.05). The expression of p-p65 and IkBε was significantly reduced in RPMI-8226 cells transfected with shRNA-REGγ-1/shRNA-REGγ-2. The degradation of IkBε was significantly lower in RPMI-8226 cells transfected with shRNA-REGγ-1 than the control (longer half-life period). Besides, the expression of MMP2, Bcl-xL and XIAP in RPMI-8226 cells was significantly inhibited by shRNA-REGγ-1/shRNA-REGγ-2. DISCUSSION: Knockdown of REGγ may inhibit the proliferation and migration, and promote the apoptosis of RPMI-8226 cells possibly by downregulating NF-κB signal pathway.

A dual-label time-resolved fluorescence immunoassay for the simultaneous determination of ferritin and ß2 -microglobulin.

BACKGROUND: Lymphocytic leukemia is a kind of primary malignant tumor of hematopoietic tissue. The aim was to establish a dual-label time-resolved fluorescence immunoassay (TRFIA) for the simultaneous determination of ferritin (FER) and ß2 -microglobulin (ß2 -MG) for the early screening and follow-up surveillance of lymphocytic leukemia. METHODS: The sandwich immunoassay was used to detect the concentration of FER, and the competitive immunoassay was used to detect the concentration of ß2 -MG in serum. FER in serum was captured by anti- FER antibody immobilized on microtiter wells, and then banded together with another anti- FER labeled with europium(III) Eu3+ chelate, followed by fluorescence measurement using time-resolved fluorometry (TRF). Sm3+ labeled ß2 -MG and ß2 -MG samples were added to compete with a certain amount of anti-ß2 -MG antibody, followed by fluorescence measurement using TRF. The performance of this dual-label TRFIA was evaluated using the clinical blood and compared with the commercial assays. RESULTS: The linear correlation coefficient (R2 ) of the FER and ß2 -MG standard curves were 0.9914 and 0.9927, respectively. The sensitivity for FER detection was 8 ng/mL (dynamic range 0-1000 ng/mL), the average recovery was 100.51%; The sensitivity for ß2 -MG detection was 1 ng/mL (dynamic range 0-1000 ng/mL), the average recovery was 101.02%. High correlation coefficients (R2 ) were obtained between the commercial assays (R2 =.9966 for FER, and R2 =.9897 for ß2 -MG). CONCLUSION: The present dual-label TRFIA has high sensitivity, specificity, and accuracy in clinical sample analysis. It is an effective detection method for the early screening and follow-up surveillance of the acute and chronic lymphocytic leukemia.

Anti-CD19 chimeric antigen receptor T-cell therapy for adult Philadelphia chromosome-positive acute lymphoblastic leukemia: Two case reports.

RATIONALE: The presence of the Philadelphia chromosome (Ph) in acute lymphoblastic leukemia (ALL) has been associated with a high risk of disease relapse and a poor prognosis. Allogeneic hematopoietic stem cell transplantation (HSCT) is an established treatment for adults with Ph-positive ALL, but relapse remains the primary cause of treatment failure, and is associated with an extremely poor prognosis. The emergence of resistance to tyrosine kinase inhibitors (TKIs) poses a challenge for patients with disease relapses after initial treatment with TKI-containing regimens. PATIENT CONCERNS: Two patients with TKI-resistant recurrent Ph-positive ALL. DIAGNOSES: Ph-positive ALL. INTERVENTIONS: Anti-CD19 CAR T-cell infusion. OUTCOMES: One patient's bone marrow blasts decreased significantly, and the other reached negative minimal residual disease (MRD). However, we first recorded the development of new-onset acute graft-versus-host disease (aGVHD) after anti-CD19 CAR T-cell infusion in a patient who received allogeneic HSCT. Our 2 case reports also demonstrate the efficacy of anti-CD19 CAR T-cell therapy in the treatment of TKI-resistant Ph-positive ALL. LESSONS: Our report suggests that anti-CD19 CAR T-cell therapy may be a promising option for the treatment of relapsed Ph-positive ALL after conventional chemotherapy or allogeneic HSCT. However, caution is due given the possibility of the adverse effects of cytokine release syndrome (CRS)-induced aGVHD for patients receiving allogeneic HSCT.

[Allogeneic hematopoietic stem cell transplantation for the treatment of mucopolysaccharidosis type 1: a case report].

Mucopolysaccharidosis type I (MPS-I) is an inborn error of metabolism with progressive multisystem involvement. Hurler syndrome is the most severe form of MPS-I that causes progressive deterioration of the central nervous system with ensuing death. This study reported the therapeutic effect of allogeneic hematopoietic stem cell transplantation (allo-HSCT) on Hurler syndrome in one case. The patient was a 25-month-old boy. He underwent allo-HSCT. The donor was his elder sister whose HLA-B locus was not matching. The reduced-intensity of BuCy conditioning regimen in allo-HSCT for this patient was as follows: busulfan 3.7 mg/kg daily at 9 to 6 days before transplantation, cyclophosphamide 42.8 mg/kg daily at 5 to 2 days before transplantation, and rabbit antithymocyte globulin 3.5 mg/kg daily at 1, 3, 5, and 7 days before transplantation. Human granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells (CD34+ cells 12.8 x10(6)/kg) were infused and cyclosporine (CSA), short-course methotrexate, daclizumab and mycophenolate mofetil (MMF) were administered to prevent graft-versus-host disease (GVHD). Complete donor-type engraftment was confirmed by Short Tandem Repeat-Polymerase Chain Reaction (STR-PCR) on day 14 after transplantation. Neutrophil and platelet engraftment occurred on days 11 and 19 after transplantation respectively. Only grade I regimen-related toxicity of live and gastrointestinal tract occurred. GVHD and graft failure were not observed. After transplantation, the clinical symptoms and the neurocognitive function were greatly improved in this patient. It was concluded that allo-HSCT was effective for the treatment of MPS-I. The reduced-intensity conditioning regimen was helpful to decrease the regimen-related toxicity. Sufficient immunosuppressive therapy and adequate hematopoietic stem cells infusion may be beneficial to the donor cell engraftment and reducing the incidence of graft failure and GVHD.

[Relationship between tumor necrosis factor genetic polymorphisms and acute lymphocytic leukemia].

BACKGROUND & OBJECTIVE: Acute lymphocytic leukemia(ALL), which is sensitive to tumor necrosis factor (TNF) is one of hematological malignances derived from lymphoid tissue. Genetic polymorphisms in the tumor necrosis factor (TNF) locus can affect transcription and expression of TNF genes. This study was designed to investigate the relationship between -308 bp polymorphism in tumor necrosis factor-alpha(TNFalpha) gene and +252 bp in lymphotoxin-alpha(LTalpha) gene and the pathogenesis, clinical course, and outcome of ALL. METHODS: The single base mutation polymorphism in TNFalpha gene and LTalpha gene were analyzed among 29 Chinese patients with ALL and 72 normal controls using polymerase chain reaction (PCR)-restrictive fragment length polymorphism (RFLP). The clinical data were collected and survival analysis was performed. RESULTS: The difference of distribution of genotypes, alleles of TNFalpha(-308), LTalpha(+252), and TNF/LT polymorphic extended haplotypes between the ALL patients and control group were not statistically significant (P >0.05). In patients, no statistically significant association was found between the presence of a given TNF/LT haplotype status and clinical characters such as sex, white blood cell (WBC) counts, central nervous system involvement, and the response to therapy(P >0.05). The estimated 1-year overall survival rates in the groups of patients carrying high-risk and low-risk haplotypes were not statistically significant (P >0.05) using Kaplan-Meier method. In multivariate Cox regression models the TNF/LT haplotype status was not found to be a risk factor for outcome (P >0.05). CONCLUSION: These data suggest that genetic polymorphisms in the TNFalpha(-308) and LTalpha(+252) are not crucial in the pathogenesis, clinical course, and outcome of ALL patients.